1. HPV 16 and 18 E6/E7 RNAs are efficiently spliced: The E6 and E7 genes of high-risk oncogenic HPV 16 and 18 are two major viral oncogenes which contain an intron in the E6 coding region. The HPV16 E6 intron is more complex than the HPV18 E6 intron since the 16E6 intron has one 5' splice site (ss) at nt 226 and two alternative 3' ss, respectively at nt 409 and nt 526. We have shown that transcripts of unspliced, full-length E6 are in extremely low abundance in several HPV 16+ and HPV18+ cervical cancer cell lines. In vitro splicing of the HPV16 E6/E7 pre-mRNA demonstrated that it was spliced efficiently only when capped, as splicing reactions could be inhibited by adding excess m7GpppG cap analogues, implying that RNA 5' capping machinery promotes recognition of a cap-proximal nt 226 5' ss. Splicing efficiency and gene product distribution was also examined in vivo. The E6/E7 coding region cloned upstream of EGFP but immediately downstream of a CMV promoter, resembles its native position in the virus genome, with the cap-proximal exon of 159 nts, and when transcribed had its pre-mRNAs spliced efficiently. When cloned downstream of EGFP, however, the cap-proximal E6/E7 nt 226 5' ss had a distance of approximately 900 nts from the cap and was spliced poorly. The resulting unspliced E6 RNA was exported to the cytoplasm, its protein product of this particular E6 mRNA was imported into nucleus and accumulated in nucleoli. The subcellular distribution of unspliced E6 protein differs from that of spliced E6*I or *II which localizes to the nucleus but not within nucleoli, indicating a difference in protein behavior. Regardless, size limiting studies on cap-proximal exon architecture demonstrate that an optimal cap-proximal exon size, smaller than 300 nts, is helpful for an efficient splicing of both the E6/E7 pre-mRNAs and human beta-globin pre-mRNAs. Collectively, our data indicate that spacing between cap structure and cap-proximal 5' ss is a limiting factor for the capping-dependent RNA splicing. In addition, we have mapped nuclear localization signals of HPV16 E6 to two regions enriched in basic amino acids. Mutational analyses are underway to characterize those NLS in different systems. 2. Cellular splicing factors involved in viral RNA splicing: Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5' splice site (ss) alternatively splices to a proximal 3' ss (at nt 3225) to express L2 or to a distal 3' ss (at nt 3605) to express L1. Two exonic splicing enhancers (ESE), each containing two ASF/SF2 binding sites, are located between the two 3' ss and have been identified as regulating the alternative 3' ss usage. The present report demonstrates that ASF/SF2 is required for the expression of BPV-1 late RNAs and for selection of the proximal 3' ss for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline (tet)-repressible promoter. Depletion of ASF/SF2 from the cells by tet greatly decreased viral RNA expression and RNA splicing at the proximal 3' ss, while increasing use of the distal 3' ss in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-IgM-BCR cross-linking rescued the viral RNA expression and splicing at the proximal 3' ss, and enhanced Akt phosphorylation and expression of the phosphorylated SR proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific PI3K/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3' ss, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the PI3K/Akt pathway and is associated with the enhanced expression of other SR proteins. 3. Structural and functional analyses of BPV-1 late stage-specific exonic splicing enhancer SE4. We have recently found a late stage-specific splicing regulatory element designated as SE4. The SE4 is A/C-rich element that is positioned downstream of a late stage-specific 3' splice site, nt 3605 3' splice site and is required for selection of the nt 3605 3' splice site at the late stage of BPV-1 infection. The SE4 consists of three ACC repeats (ACCACCACC) and mutations in the first and last ACC, not the middle ACC, abolish the SE4's function both in vitro and in vivo splicing assays. Currently, we are trying to investigate cellular/viral proteins responsible for the function of the SE4 by using RNA-protein interaction and immunoprecipitation techniques. 4. RNA splicing of KSHV K8 and K8.1. Kaposi's sarcoma-associated herpesvirus (KSHV) K8 and K8.1 ORFs are juxtaposed and span from nt 74850 to nt 76695 of the virus genome. A K8 pre-mRNA overlaps the entire K8.1 coding region and alternative splicing of KSHV K8 and K8.1 pre-mRNAs each produces three isoforms (a, b and g) of the mRNAs. We have mapped the 5' end of the K8.1 RNA in butyrate-induced KSHV-positive JSC-1 cells to nt 75901 in KSHV genome and shown that exon 3 of the K8 pre-mRNA in JSC-1 cells covers most part of the intron 3 previously defined and has three 5' splice sites (ss), respectively, at nt 75838, nt 76155, and nt 76338. Selection of the nt 75838 5' ss dictates the K8 mRNA production and overwhelms the RNA processing. Alternative selection of other two 5' ss is feasible and leads to production of two additional bicistronic mRNAs: K8/K8.1a and b. However, the novel bicistronic K8/K8.1 mRNAs translated a little K8 and no detectable K8.1 proteins in 293 cells. Data suggest that production of the K8/K8.1 mRNAs may be an essential way to control K8 mRNAs, especially K8a, to a threshold at RNA processing level. Works are in progress in characterizing suboptimal features of the K8 intron 2 that are likely responsible for producing K8 b. 5. Identification of KSHV K8.1 late promoter. After we mapped transcription start site of the late K8.1 to nt 75901 in the virus genome, we proposed presence of a potential K8.1 later promoter upstream of the start site. To identify such a putative late promoter, a dual luciferase system has been developed in JSC-1 cells, a KSHV+ B cell line, for analysis of the putative promoter activities since viral late promoter activities of many herpesviruses usually require viral DNA replication. In this system, a putative K8.l promoter with various sizes was cloned upstream of luciferase gene and the activities of the resulting plasmids were examined in JSC-1 cells along with or without butyrate induction, a common means to activate viral late gene expression and KSHV lytic cycle in KSHV+ B cells. Our preliminary results show that the putative promoter had no activity in uninduced JSC-1 cells, but its activities could be greatly induced by butyrate in JSC-1 cells, not in other cell lines such as 293 and Raji cells (EBV+, KSHV-), indicating that the activities of the putative K8.1 late promoter are JSC-1 cell-specific. Extensive mapping has identified a promoter core with approximately 23 bp in length. The core element has a 5' CG-rich region and a 3' TA-rich region. The activities of the core in butyrate-induced JSC-1 cells are orientation-dependent and depend on the presence of the 5' CG region. More interestingly, the activities of the putative promoter in butyrate-induced JSC-1 cells could be inhibited by PAA, a viral DNA polymerase inhibitor, approximately 70%, suggesting that the activities of the putative K8.1 promoter is likely depended on the viral DNA replication. Data indicate that KSHV initiates its gene expression of ORF50 (immediate early), K8 (early) and K8.1 (late) clustered at one locus in the virus genome through alternative promoter switch at different stages of the virus life cycle. Further studies are under way with newly established RNA interference knockout techniques to strength the linkage of the late promoter activities to the viral DNA replication.